GSM7049136: Psoriasis skin 3; Homo sapiens; RNA-Seq
Skin samples were frozen in OCT medium and stored at -80°C until sectioning. Optimization of tissue permeabilization was performed on 20-μm-thick sections using Visium Spatial Tissue Optimization Reagents Kit (10X Genomics, Pleasanton, CA, USA), which established an optimal permeabilization time to be 24 minutes. Samples were mounted onto a Gene Expression slide (10X Genomics), fixed in ice-cold methanol, stained with hematoxylin and eosin, and scanned under a microscope (Keyence, Itasca, IL, USA). Tissue permeabilization was performed to release the poly-A mRNA for capture by the poly(dT) primers that are precoated on the slide and include an Illumina TruSeq Read, spatial barcode, and unique molecular identifier (UMI). Visium Spatial Gene Expression Reagent Kit (10X Genomics) was used for reverse transcription to produce spatially barcoded full-length cDNA and for second strand synthesis followed by denaturation to allow a transfer of the cDNA from the slide into a tube for amplification and library construction.   For Visium for FFPE, the skin samples were formalin-fixed and paraffin-embedded. 5-micron thick sections were mounted onto a Visium Gene Expression slide (10X Genomics), baked at 42°C for 3 hours, and dried in a desiccator at room temperature overnight. For deparaffinization, the slide was incubated at 60°C for 2 hours, immersed in xylene, and rehydrated in an ethanol gradient. H & E staining was then performed using Mayer's hematoxylin (Millipore Sigma), bluing reagent (Dako, Agilent), and alcoholic eosin (Millipore Sigma). Stained slides were scanned under a microscope, followed by decrosslinking using 0.1 N HCl and TE Buffer (pH 9.0) to release RNA that was sequestered by formalin. Human whole transcriptome probe panel (10x) that consisted of a pair of specific probes (5' containing Small RNA Read 2S and 3' containing poly-A) for each gene was hybridized to RNA. Probe pairs were then ligated to seal the junctions between them and to form the single-stranded ligation products. The samples were treated with RNase and permeabilized to release the ligation products. Poly-A portion of the products was then captured by the poly(dT) regions of the capture probes precoated on the Visium slide that also include an Illumina Read 1, spatial barcode, and unique molecular identifier (UMI). Probes were extended to produce spatially barcoded ligated probe products and released from the slide for indexing via Sample Index PCR and final library construction and sequencing. Visium Spatial Single Cell 3ʹ Gene Expression libraries consisting of Illumina paired-end sequences flanked with P5/P7 were constructed after enzymatic fragmentation, size selection, end repair, A-tailing, adaptor ligation, and PCR. Dual Index Kit TT Set A (10X Genomics) was used to add unique i7 and i5 sample indexes and generate TruSeq Read 1 for sequencing the spatial barcode and UMI and TruSeq Read 2 for sequencing the cDNA insert, respectively.